In the oral environment, interactions between lectin-like adhesions on oral bacteria and complementary glycan recognition motifs on oral host cell and tooth surfaces and on the surfaces of other oral bacteria play important roles for initial bacterial colonization and the subsequent formation of multi-species biofilms that have the potential to turn into pathogenic habitats causing dental, oral, and perhaps systemic disease. Considering (a) the multitude of bacterial species identified in oral biofilms, (b) the rich diversty of glycans found on salivary glycoproteins, and (c) the innumerable glycan motifs expressed on the surface of other bacteria, it is likely that many more unidentified lectin-like bacterial adhesns exist in the oral microbiome, beyond the few ones currently known. Once identified, those bacterial lectin-like adhesins, many of which presumably will exhibit novel glycan-binding specificities, can be forged by recombinant DNA techniques into highly specific tools for the detecting and localization of corresponding glycan receptor structures. The overall objective of this application is to mine the oral microbiomes of humans and other mammals for novel glycan- binding bacterial adhesions by using glycan-functionalized targeted probes. Here, we focus on one well-characterized class of bacterial adhesions, the serine-rich repeat (SRR) proteins of Gram- positive bacteria. By identifying a novel set of glycan-binding proteins with unique ligand-binding properties, we aim to demonstrate as a proof of principle that it will become feasible to expand the range of currently available glycan-binding probes enormously. The specific aims of the project are to 1. Isolate SRR adhesion-bearing bacteria from the oral cavity of humans and other mammalian species that bind specifically to host sialoglycans containing 4- or 9-O-acetylated forms of N-acetyl and N-glycolylneuraminic acid. 2. Isolate SRR adhesin-bearing bacteria from human oral biofilms that bind specifically to bacterial polysaccharide receptors containing the unusual lectin-recognition motifs (Rha1-2Rha and Gal1- 6Glc). 3. Use bioinformatics to identify novel glycan-binding regions in SRR proteins, and use bacterial genomics to generate recombinant glycan-binding probes for detection of a diverse set of glycans.